Affinity chromatography matrix

Abstract

The present invention relates to a method of separating one or more immunoglobulin containing proteins from a liquid. The method includes first contacting the liquid with a separation matrix comprising ligands immobilised to a support; allowing the immunoglobulin containing proteins to adsorb to the matrix by interaction with the ligands; followed by an optional step of washing the adsorbed immunoglobulin containing proteins; and recovering said immunoglobulin containing proteins by contacting the matrix with an eluent which releases the proteins. The method improves upon previous separation methods being that each of the ligands consists essentially of a monomer or dimer SpA or protein Z or a functional variant thereof.

1 . In a method of separating one or more immunoglobulin containing proteins from a liquid, which method comprises
 (a) contacting the liquid with a separation matrix comprising ligands immobilised to a support;   (b) allowing said immunoglobulin containing proteins to adsorb to the matrix by interaction with the ligands;   (c) optionally washing the adsorbed immunoglobulin containing proteins; and   (d) recovering said immunoglobulin containing proteins by contacting the matrix with an eluent which releases the proteins;   the improvement comprising that each of said ligands consists essentially of a monomer or a dimer of domain B of staphylococcal Protein A (SpA), protein Z, or a functional variant thereof.   
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1 , wherein the ligands are alkali-stable by mutating at least one asparagine residue to an amino acid other than glutamine. 
     
     
         4 . The method of  claim 3 , wherein the ligands have affinity for the Fc part of an immunoglobulin but lack affinity for the Fab part of an immunoglobulin. 
     
     
         5 . The method of  claim 4 , wherein at least one glycine of said ligands has been replaced by an alanine. 
     
     
         6 . The method of  claim 4 , wherein a glycine residue at position 29 has been changed to an alanine. 
     
     
         7 . The method of  claim 1 , wherein the ligand is Protein Z in which the alkali-stability has been achieved by mutating at least one asparagine residue to an amino acid other than glutamine. 
     
     
         8 . The method of  claim 7 , wherein the alkali-stability of Protein Z has been achieved by mutating at least the asparagine residue at position 23 to an amino acid other than glutamine. 
     
     
         9 . The method of  claim 1 , wherein the recovery of immunoglobulin containing proteins is achieved by adding an eluent having a pH of 3.8-4.2. 
     
     
         10 . The method of  claim 1 , wherein at least 80%, such as at least 90%, and preferably at least 95% of the immunoglobulin containing proteins are recovered using an eluent having a pH of 3.8-4.2. 
     
     
         11 . The method of  claim 1 , wherein the recovery of immunoglobulin containing proteins is achieved by adding an eluent having a pH of 4.0-4.4. 
     
     
         12 . The method of  claim 1 , wherein at least 80%, such as at least 90%, and preferably at least 95% of the immunoglobulin containing proteins are recovered using an eluent having a pH of 4.0-4.4. 
     
     
         13 . The method of  claim 1 , wherein the immunoglobulin containing protein is a monoclonal antibody. 
     
     
         14 . The method of  claim 1 , wherein the immunoglobulin containing protein is a polyclonal antibody. 
     
     
         15 . The method of  claim 1 , wherein the immunoglobulin containing is a fusion protein containing an immunoglobulin containing fused with another protein.